This year we have again been involved in many projects relating to the analysis of peptides and proteins that have been brought to our attention by researchers at NHLBI and NIH. The goal is to use advanced mass spectrometric technology to study the biology of a particular system by obtaining information on the identity of proteins either through mass spectrometric sequencing or by simply carefully measuring the masses of a sufficient number of its peptides. This technique is unique in identifying post-translational modifications (PTMs) essential for protein/cellular functions. With the state-of-the-art Micromass QTOF3 mass spectrometer and other LC-MS and MALDI-TOF instruments, we were able to perform many experiments on biological samples, mostly in this protein and peptide class. For instance, we have identified several immunoprecipitated proteins that interact specifically with many Arf family members such as AGAP and ARAP-1. Proteins that bind and thus play an important role in regulating the activity of methionine sulfoxide reductase have been identified. We started cataloging proteins from nuclear matrix as well as soluble preparations from human Jurkat cells using high throughput robotic digestion system to perform tryptic digestion, followed by LC-MS/MS. We have also worked with a group from Johns Hopkins University identifying proteins that interact specifically with GB3, a sphingolipid highly expressed in colon cancer Caco-2 cells. In the area of PTMs we have studied glutathionylation of HIV protease via cysteine residues that modulate its enzymatic activity, disulfide linkages in recombinant translin which is very important in oligomerization upon oligonucleotide binding and in human glutathione peroxidase 5, tyrosine phosphorylation of ARAP-1 coexpressed with Src, possible N-terminal truncation of mouse ZP2 (zona pellucida glycoprotein 2) important in egg polyspermy prevention and in the N- and O-glycosylation site determination of human ZP3. We have spent a significant amount of time optimizing sensitivity and resolution of our QTOF3, its capillary LC and its communication with the mass spectrometer. We also performed some experiments on labeling proteins with commercially available ICAT technology for protein quantitation which should have considerable application with our newly-purchased 2D-LC ProteomeX system from ThermoFinnigan.